The goal of the proposed research is to develop an improved approach for the treatment of blood cancers that utilizes human enzymes rather than bacterial enzymes. Currently, a mainstay treatment of such cancers includes the administration of a bacterial enzyme called asparaginase. The activity of this enzyme is to hydrolyze the amino acid asparagine to aspartic acid and ammonia. Certain blood cancers are dependent on the extracellular pool of asparagine. Administration of asparaginase depletes this source, depriving the cancer cells of a vital amino acid, and ultimately induces cell death (apoptosis). Because all currently approved preparations of asparaginases are of bacterial origin, a substantial proportion of patients have an immune response against the enzyme - a reaction that can be deadly. Moreover, the generated anti-bacterial asparaginase antibodies clear the enzyme from circulation, thereby eliminating its anticancer potential and preventing further administration of the drug. A further complication is due to the undesired glutaminase activity of bacterial asparaginases, which is a source of toxicity of this treatment. We propose to replace the bacterial enzymes with human asparaginases. This will abolish the immune response. We will also engineer the human enzymes to be devoid of glutaminase activity, thereby eliminating this cause of toxicity. The wild-type versions of human asparaginases are not suitable for replacing the bacterial enzymes since their Km value is in the millimolar range, yet the concentration of asparagine in blood is only about 50 micromolar. Indeed, the bacterial enzymes used in the clinic, from E. coli and Erwinia, have a low Km value for asparagine. The research in this proposal delineates a strategy to engineer human asparaginases to have this vital property of low Km with asparagine. In Aim 1 we will study the structure/function properties of two human asparaginases. We will incorporate structural, biochemical, kinetic, and mutagenesis approaches in this part of the proposal. The data from Aim 1 will inform the engineering studies of Aim 2. The strategy is to introduce mutations that result in (i) lowering of the asparagine Km value to the micromolar range, (ii) an enzyme devoid of glutaminase activity, and (iii) improved thermo-stability. The latter is to increase the circulation half-life of the enzyme in the patientsso that the asparaginase activity is long lasting. An additional novel aspect of Aim 2 is the linking f the human asparaginase to human serum albumin (HSA). Since HSA has a circulation half-life of ~20 days, we hypothesize that the fusion HSA- asparaginase will have increased circulation half-life relative to the free enzyme. This will significantly aid the clinical use of this drug, ad will result in more persistent asparagine depletion. Aim 3 will test the cell-killing power of the engineered asparaginases in cell culture, and the stability of the enzymes in plasma. The purpose of Aim 3 is to ready the engineered human asparaginases for clinical trials. Adults treated with the bacterial asparaginases exhibit a more intense immune response compared to pediatric patients. Hence, the less immunogenic enzymes developed here will especially be beneficial to this patient population. This makes this proposal especially relevant to the treatment of veterans.